Capsular polysaccharide adhesin antigen, preparation, purification and use

ABSTRACT

A substantially pure capsular exopolysaccharide adhesin of coagulase-negative staphylococcal strains, and a general method to prepare such adhesins, are described. Vaccines composed of such adhesins, and uses of such adhesins to produce polyclonal and monoclonal antibodies against such adhesins, are also disclosed. The adhesins are useful in coating polymeric medical materials to prevent colonization by coagulase-negative staphylococcal strains, and as a probe in selecting desirable polymeric medical materials. Such adhesin antibodies are useful in vivo to prevent infection by nosocomial coagulase-negative staphylococcal strains, in assays for the detection of such bacteria, in assays for the estimation of such adhesins in complex mixtures, and as an affinity chromatography matrix.

This application is a divisional of U.S. patent application Ser. No.08/336,688, filed on Nov. 7, 1994, now U.S. Pat. No. 5,980,910 which isa continuation of U.S. patent application Ser. No. 08/033,756 (nowabandoned), filed on Mar. 18, 1993, which is a continuation of U.S.patent application Ser. No. 07/727,982 (now abandoned), filed on Jul.10, 1991, which is a divisional of U.S. patent application Ser. No.07/250,417, filed Sep. 28, 1988 which is now U.S. Pat. No. 5,055,455.The entire contents of all the foregoing issued patents and patentapplications are incorporated by reference herein.

GOVERNMENT SUPPORT

Some aspects of the present invention were made with support by grantsfrom the United States National Institutes of Health (NIH) under NIHcontract No. R01-AI23335. The U.S. Government may retain certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to a substantially pure exopolysaccharideadhesin isolated from a particular strain of Staphylococcus epidermidis,to a general method capable of isolating this compound in substantiallypure form, and to uses of said purified adhesin product as a vaccine forthe production of antibodies effective against the binding of homologousbacterial cells to polymeric materials, and as a probe for thedevelopment of polymeric materials useful as catheters and medicalprostheses.

DESCRIPTION OF THE BACKGROUND ART

Both Staphylococcus aureus (coagulase-positive) and Staphylococcusepidermidis (coagulase-negative) have a characteristic propensity forinvading skin and adjacent tissues at the site of prosthetic medicaldevices, including intravascular catheters, cerebrospinal fluid shunts,hemodialysis shunts, vascular grafts, and extended-wear contact lenses.Within 48 to 72 hours, relatively large numbers of staphylococci aredemonstrable at the site of insertion of these foreign bodies. Archer,G. L., “Staphylococcus epidermidis: The Organism, Its Diseases, andTreatment,” in Remington, J. S., et al., eds., Current Clinical Topicsin Infectious Diseases, McGraw-Hill, New York, 1986, pp. 25-46; Youmans,G. P., et al., The Biologic and Clinical Basis of Infectious Diseases,Saunders, Philadelphia, 1985, pp. 618-625, 738-9. It has beendemonstrated that S. epidermidis cells attach and proliferate on theinner or outer surfaces of catheters, irrespective of their composition(polyethylene, polyvinylchloride, polyvinylfluoride, or polyester basedmaterials).

Although the virulence of coagulase-negative staphylococci clearly isenhanced in the presence of a foreign body, the microbial factor(s) thatpermit these normal skin commensals to become nosocomial pathogens havenot been well characterized. As adherence is believed to be the criticalfirst step in the pathogenesis of coagulase-negative staphylococcalforeign-body infections, attention has focused on surface properties ofthese organisms that might mediate adherence to, and colonization of,polymeric prosthetic materials.

The most promising candidate for the source of a specific staphylococcaladhesin is an extracellular material often referred to as “slime.” Ithas been hypothesized that the slime substance may protect the S.epidermidis cells against antibiotics, as well as against natural hostdefense mechanisms. Youmans et al., supra; Peters, G., et al., Journalof Infectious Diseases 146:479-82 (1982).

It has been known since 1972 that coagulase-negative bacteria isolatedfrom cerebrospinal fluid shunt infections elaborate a mucoid materialthat stains with alcian blue and is presumably a polysaccharide.Bayston, R., et al., Developmental and Medical Child Neurology 14 (Supp.27):25-8 (1972). The extracellular polysaccharide substance ofslime-producing bacteria is a loose amorphous material composed of arange of low and high molecular weight polymers composed, in general, ofneutral monosaccharides such as D-glucose, D-galactose, D-mannose,L-fucose, and L-rhamnose, and also contain amino sugars, uronic acid,and polyols such as ribitol and glycerol. Gristina, A. G., Science237:1588-95 (1987). Glucose, galactose, phenylalanine, mannose,hexosamine, phosphorous, glycine and alanine have been found ascomponents of the slime produced by S. epidermidis strains in clinicalspecimens unrelated to biomaterial infections. Ichiman, J., et al., J.Appl. Bact. 51:229 (1981). Isolates of such bacteria from sites ofinfections are more likely to produce slime than are random isolatesfrom skin. Ishak, M. A., et al., Journal of Clinical Microbiology22:1025-9 (1985). Moreover, slime-producing strains adhere well to avariety of polymeric materials. Christensen, G. D., et al., Infect.Immun. 37:318-26 (1982).

Coagulase-positive staphylococci (S. aureus) are reported to producemultiple cell surface proteins which can be released from such cells bythermal extraction and which can be shown to bind to influenzavirus-infected canine kidney cells. It was considered that S. aureusproduces multiple cell surface protein adhesins. Sanford, B. A., et al.,Infect. Immun. 52:671-5 (1986); Proc. Soc. Exp. Biol. Med. 181:104-11(1986).

Identification of other microbial adhesins has been reported. Pier (U.S.Pat. No. 4,285,936, Aug. 25, 1981; U.S. Pat. No. 4,528,458, Mar. 25,1986) discloses a method for partial purification of a polysaccharideantigen from Pseudomonas aeruginosa slime. Escherichia coli fimbrialprotein adhesins have been identified and partially purified by severalinvestigators (Orskov, I., et al., Infect. Immun. 47:191-200 (1985);Chanter, H., J. Gen. Microbiol. 12:225-243 (1983); Ferreiros, C. M., etal., Rev. espanol. de fisiolog. 39:45-50 (1983); and Moch, T., et al.,Proc. Natl. Acad. Sci. 84:3462-6 (1987)).

Lectin-like glycoprotein adhesins have been identified in theBacteroides fragillis group, and a 70 kDa adhesin has been purified byaffinity chromatography (Rogemond, V., et al., Infect. Immun. 53:99-102(1986)). Monoclonal antibody affinity chromatography was used to purifya 165 kDa surface protein of Mycoplasma pneumoniae which mediatesattachment of such bacteria to target cells (Leigt, D. K., et al., J.Bacteriol. 157:678-80 (1984)), and to isolate a 150 kDa adhesin proteinfrom Streptococcus sanguis FW213 (Elder, B. L., et al., Infect. Immun.54:421-7 (1986)). A uroepithelial cell adhesin protein of 17.5 kDa waspartially purified from fimbrii of Proteus mirabilis, a frequent causeof urinary tract infection (Wray, S. K., et al., Infect. Immun. 54:43-9(1986)).

Ludwicka (Ludwicka, A., et al., Zbl. Bakt. Hyg. A 258:256-67 (1984))fractionated by ion-exchange chromatography a phenol-saline extract ofslime from S. epidermidis and obtained four crude fractions. Both thephenol-saline extract and two of the four crude fractions inhibited theattachment of bacterial cells to polymeric material. On the basis of thepresence of monosaccharides in the fractions, the reaction of thefractions with lectins, and the complete inhibition of the production ofthe four fractions by pretreatment of the bacteria by tunicamycin(inhibitor of glycoprotein synthesis), the authors concluded that theextracellular slime substance is a complex of glycoconjugate (i.e.,glycoprotein) character.

Hogt (Hogt, A. H., et al., Infect. Immun. 51:294 (1986) have alsoobserved that crude extracellular products from the slime of homologousstrains of S. epidermidis inhibit the adherence of homologous bacterialcells to polymeric materials used as catheters and prostheses. Noinformation was provided in this report as to the chemical nature of theextracellular products.

Bacterial cells and materials derived from the surface of such cellshave been used as vaccines to produce antibodies directed againsthomologous bacteria. Frank (Frank, R., etal., French Patent Application85-07315, published Nov. 21, 1986) discloses a covalent conjugatebetween a capsular protein adhesin (MW-74 kDa) from Streptococcus mutansand a polysaccharide from the same (serotypically) organism, and the useof said conjugate as an anti-caries vaccine, Pier (Pier, G. B., et al.,U.S. patents, supra) disclose a vaccine comprising a high molecularweight mucoid exopolysaccharide from Pseudomonas aeruginosa strain 2192slime which induces in recipient animals an immunity to said organism.Sadowski (Sadowski, P., U.S. Pat. No. 4,443,549, Apr. 17, 1984; U.S.Pat. No. 4,652,498, Mar. 24, 1984; and EP 82401506.1, published Apr. 27,1983) discloses monoclonal antibodies specific for surface adhesins ofEscherichia coli and Pseudomonas aeruginosa which may be used for thetherapeutic treatment of diseases induced by adhesin-bearing homologousbacteria in humans and animals. Nagy (Nagy, L. K., et al., Dev. Biol.Stand. 53:189-97 (1983)) discloses multi-adhesin vaccines for theprotection of the neonatal piglet against ‘Escherichia coli infections.

SUMMARY OF THE INVENTION

The inventors considered that, if a substantially pure capsularpolysaccharide adhesin antigen could be isolated from the slime ofstrains of pathogenic S. epidermidis, a vaccine could be prepared fromsuch an antigen that could be used to raise polyclonal antibodies invivo in a human or animal, or monoclonal antibodies in hybridoma cells.Reasoning that adhesin-mediated colonization is required for the onsetof pathogenesis, the inventors conceived that the polyclonal ormonoclonal antibodies produced against the adhesin of the invention, bypreventing the adherence of adhesin-bearing pathogenic bacteria to therecipient's tissue cells or polymeric medical prostheses or catheters,represent a new means for preventing or treating diseases and infectionsdue to S. epidermidis.

Further, the substantially pure capsular polysaccharide adhesin of theinvention is useful as a probe to test new polymeric materials formedical devices.

Therefore, in a preferred embodiment, the present invention provides asubstantially pure polysaccharide from extracts of S. epidermidis RP-62strain (an isolate from a patient with catheterrelated bacteremia thatproduces copious quantities of slime) that mediates adherence topolymeric materials and also appears to be the capsule for thisorganism. In another preferred embodiment, the present inventionprovides a method for producing a substantially pure polysaccharideadhesin from extracts of S. epidermidis strain RP-62.

In another preferred embodiment, the substantially pure polysaccharideadhesin of the invention is used as a vaccine to raise in animalsantibodies against said adhesin that inhibit the attachment ofadhesin-bearing bacteria to polymeric materials.

The substantially pure polysaccharide of the invention may also be usedas an antigen to produce monoclonal antibodies in hybridoma cells. Suchmonoclonal antibodies can be administered for prophylaxis or therapeuticpurposes to humans or animals in order to prevent or reduce infectionsby coagulase-negative staphylococci.

In yet another preferred embodiment, the substantially purepolysaccharide adhesin of the invention is used to screen polymericmaterials for resistance to attachment by bacteria.

DESCRIPTION OF THE DRAWINGS

FIG. 1A demonstrates the immunodiffusion pattern of crude extract (A),purified teichoic acid (B) and purified adhesin (C) against antiseraraised to whole cells of S. epidermidis strain RP-62A.

FIG. 1B demonstrates immunoelectrophoresis of S. epidermidis antigens.Troughs were filled with antisera to strain RP-62A whole cells. A, crudeextract; B, teichoic acid, C, purified adhesin; D, mixture of teichoicacid and purified adhesin.

FIG. 2 demonstrates the electrophoresis pattern of restriction enzymedigests of bacterial DNA from strains RP-62A (left-hand pattern of eachpair) and RP-62NA (right-hand pattern of each pair). Lanes 1 and 12,HindIII digest of phage lambda DNA; Lanes 2 and 3, undigested DNA fromRP-62A and RP-62NA; Lanes 4 and 5, EcoRI digest; Lanes 6 and 7, SauIIIAdigest; Lanes 8 and 9, RsaI digest; Lanes 10 and 11, ClaI digest.

FIG. 3 demonstrates the inhibition of binding of S. epidermidis strainRP-62 cells to silastic catheter tubing after incubation of the tubingin the indicated concentrations of the various bacterial antigens priorto dipping in bacterial suspension (10⁶ cells per ml). Significant(p<0.05, t test) inhibition was seen only with crude extracts fromstrain RP-62A at concentrations of 0.12-0.50 mg/ml and with purifiedadhesin at concentrations of 0.06-0.50 mg/ml.

FIG. 4 demonstrates the inhibition of adherence of various strains ofcoagulase-negative staphylococci to silastic catheter tubing bydifferent antigen preparations (0.1 mg/ml concentration) from S.epidermidis strain RP-62A. An asterisk indicates significant (p<0.05, ttest) inhibition.

FIG. 5 shows transmission electron microscopy of various strains ofcoagulase-negative staphylococci following incubation with normal rabbitserum, rabbit serum raised to either whole RP-62A cells or rat antiserumraised to purified adhesin and ferritin-labeled goat antibody to rabbitor rat IgG. A) is strain RP-62 stained with normal rabbit serum(×75,000); B) strain RP-62 stained with rabbit antisera to whole cells(×62,000); C) strain RP-62A stained with rat antiserum to purifiedadhesin (×48,000); D) strain RP-14 stained with rabbit antiserum tostrain RP-62A whole cells (×35,000); E) strain RP-14 stained with ratantisorum to purifiod adhesin (×65,000); and F) strain RP-62NA stainedwith rabbit antiserum to strain RP-62 whole cells (×50,000). Bar in eachgraph represents 200 nm.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention comprises the isolation in substantially pure form of anexopolysaccharide adhesin antigen from coagulase-negative staphlococcibacteria, use of said adhesin as a vaccine to raise polyclonal andmonoclonal antibodies against said adhesin, use of said adhesin toprevent adherence of said bacteria to polymeric materials, and use ofsaid adhesin as a probe to test for polymeric materials to which saidbacteria will not adhere.

Materials and Methods

Bacterial Strains

The following strains were provided by Dr. Gordon Christensen, Memphis,Tenn., and have been described previously (Christensen, G. D., et al.,Ann. Intern. Med. 96:1-10 (1982); Infect. Immun. 37:318-26 (1982)): (a)Staphylococcus epidermidis strains RP-62A (slime-producing, highlyadherent, from a patient with catheter-related sepsis), RP-62NA (avariant of RP-62A which is less adherent and produces no slime bymacroscopic examination), and RP-12; (b) S. hominis strain RP-14; and(c) S. haemolyticus strain SP-2.

DNA Analysis of S. epidermidis Strains

Bacterial cells are lysed by the enzyme lysostaphin. The lysate isdigested with RNase A (Sigma) and RNase T₁ (Sigma) to degrade bacterialRNA, dissolved in a detergent solution such as sodium dodecyl sulfate,and the proteins digested with proteolytic enzymes such as pronase andproteinase K (Boehringer-Mannheim). DNA is extracted from the digestedcells by multiple extractions into phenol, and precipitated from thephenolic solution by the addition of ethanol at −20° C. at a finalconcentration of 60-70% alcohol. The precipitated DNA is collected bycentrifugation, washed with 70% aqueous ethanol, dried in vacuo, thendigested with restriction endonucleases (EcoRI, SauIIA, RsaI and ClaI(New England Biolabs, Beverly, Mass.)). The restriction digest iselectrophoresed on a 1% agarose gel; restriction fragments arevisualized by ethidium bromide staining.

Characterization of Crude Extracts, Purified Adhesin, and Teichoic Acid

Samples are hydrolyzed at 100°C in 6 N HCl from 4 to 48 hours prior toanalysis. Reducing carbohydrate content is detected and estimated by thephenol-sulfuric acid reaction (Dubois, M., et al., Anal. Chem. 28:350-6(1956)), proteins by a positive reaction in the Bradford dye test(Bradford, M., Anal. Biochem. 72:248-54 (1976)), nucleic acids byabsorbance at 254 nm against a DNA standard; phosphate by a positivereaction in the method of Chen (Chen, P. S., et al., Anal. Chem. 28:1266(1956)); lipids by gas-liquid chromatography against fatty acid methylesters as standards (Lee, J. C., et al., Infect. Immun., in press(1987)); and amino acids and amino sugars by an amino acid analyzer(Model 121 MB, Beckman Instruments, Inc., Fullerton, Calif.) using alithium citrate system. Monosaccharides are individually identified bygas liquid chromatography of the trimethylsilyl derivatizedmonosaccharide methyl esters (Chambers, R. C., et al., Biochem. J.125:1009-18 (1971)) in a Hewlett-Packard 5880 instrument usingsimultaneous injections of identical samples onto 25-foot capillarycolumns of RSL-310 (Alltech Associates, Deerfield, Ill.) and SP-2330(Supelco, Delfont, Pa.). The injector and initial oven temperatures are140° C., which is held for 3 minutes; a 5° C./min rise to 150° C. isthen performed, followed by a 30° C./min rise to 210° C., which is thenheld for an additional 9 minutes. The flame ionization detector ismaintained at 250° C. Samples are identified by retention times comparedto standards. Serologic analyses can be performed by double diffusionand immunoelectrophoresis methods (Ouchterlony, O., et al., InImmunochemistry, Vol. I, Blackwell, Oxford, 1978, Chapter 19).

Adherence Assays

The adherence of coagulase-negative staphylococcal strains to polymeric(i.e., silastic) catheter tubing (French 3, Jesco International Inc.,San Antonio, Tex.) is determined as follows. An overnight culture ofbacteria in tryptic soy broth is diluted to contain 10⁶ colony-formingunits (cfu)/ml. A 3 cm length of tubing fitted with a 21 gauge needleand sterilized with ethylene oxide gas is then dipped into the culturefor 15 min at room temperature. The tubing is washed in saline byvigorously agitating the tubing, as well as repeatedly drawing salinethrough the tubing with a 3 ml syringe fitted to the needle. Washing iscontinued until wash fluids contain less than 1 cfu/100 μl. This occursin about 3 separate washes. After discarding a 1 cm section of thetubing, bacteria adhering to the remaining 2 cm is quantified by rollingthe tubing over the surface of a tryptic soy agar plate in severaldirections, followed by overnight incubation at 37° C. The cfu/catheterare counted the next day. The efficiency of the transfer of bacteriafrom plastic tubing to the agar plate can be estimated byradio-labelling the organisms by including one μCi of [¹⁴C]-sodiumacetate in the preliminary overnight culture medium. The number of cfuadhering to the tubing before and after rolling on the agar plate isdetermined by liquid scintillation counting and correlated withbacterial counts obtained by plating identical samples.

Direct adherence of the purified adhesin to catheter tubing isdetermined by incubating a 0.5 cm length of tubing with a 0.5 mg/mlsolution of adhesin in 40 mM phosphate buffer, pH 7.2, for two hours at37° C., washing the tubing in phosphate-buffered saline 0.05% Tween 20,and performing a standard ELISA or RIA assay on the sensitized piece oftubing (Bryan, L. E. et al. J. Clin. Microbiol. 18:276-82 (1983)). Bythe term “ELISA” is intended an enzyme-linked immunoassay. By the term“RIA” is intended a radioimmunoassay.

Inhibition of adherence of bacteria to catheter tubing by crude extractsand purified adhesin is performed by incubating the catheter tubing insolutions of these materials for two hours at 37° C., washing the coatedtubing in sterile saline, placing it in bacterial cultures (10⁶ cfu/ml),and completing the adherence assay as described supra. When poorlyadherents strains of S. epidermidls (e.g., strains CL and SP-2) are usedin inhibition assays, the input inoculum should be increased to 10⁷cfu/ml, which increases the number of adhering bacteria as much as5-fold. Inhibition of adherence is calculated as follows:${\% \quad {inhibition}} = {100 - \frac{(100)\left( {{{no}.\quad {of}}\quad {cfu}\quad {adhering}\quad {following}\quad {adhesin}\quad {treatment}} \right)}{{{no}.\quad {of}}\quad {cfu}\quad {adhering}\quad {without}\quad {treatment}}}$

Inhibition of adherence by rabbit antibody to purified adhesin (seeinfra) is performed by incubating the bacteria with the indicatedconcentration of normal and immune serum for 2 hr at 4° C., washing thebacteria three times in tryptic soy broth, resuspending to 10⁶ cfu/mltryptic soy broth, and continuing the adherence assay as describedsupra. Inhibition of adherence is calculated as follows;${\% \quad {inhibition}} = {100 - \frac{(100)\left( {{{no}.\quad {of}}\quad {cfu}\quad {adhering}\quad {with}\quad {immune}\quad {serum}} \right)}{{{no}.\quad {of}}\quad {cfu}\quad {adhering}\quad {with}\quad {normal}\quad {serum}}}$

Inhibition data should be statistically analyzed for significance byStudents t test.

Transmission Electron Microscope

Transmission electron microscopy of S. epidermidis strains is performedas previously described (Pier, GB, J. Clin. Microbiol., 24:189-96(1986). For visualization of extracellular structures, bacterial cellsare incubated with either a 1:2 dilution of rabbit antibody to wholecells or undiluted rat antibody raised to purified adhesin (see infra),or with normal serum controls. After three saline washes, bacteria areincubated with ferritin-conjugated antibody to either rabbit or rat IgG.

Preparation of Crude Bacterial Extracts

Crude extracts are prepared by incubation of cell suspensions with theenzymes lysostaphin and lysozyme. Insoluble material is removed bysequential centrifugation and filtration through a micropore filter(0.45 μm), the filtrate is dialyzed against water, and then lyophilized(freeze-dried in vacuo at low temperature).

Isolation of Adhesin

Eighteen-hour cultures of S. epidermidis strains are subjected tothermal shock (95-100° C.) at about pH 5.0. The mixture is brought toneutral pH (preferably 6.8) and room temperature, then clarified bysequential centrifugation and passage through a micropore filter. Theclear extract is concentrated, neutralized, and the conductivity reduced(preferably to below 10 millisiemans) by repeated ultrafiltrationthrough a 10,000 dalton cut-off membrane and washing with water. Theretained concentrate, which contains macromolecules of mass greater than10,000 daltons, is then fractionated by ion-exchange chromatography atneutral pH (preferably about 7.0); a preferred system is DEAE Zeta-Prep250 cartridge (LKB Instruments, Rockville, Md.). Adhesin is eluted by0.2 M NaCl at neutral pH (preferably about 7.0), as determined by anadherence assay (infra). Adhesin-containing fractions are then subjectedto affinity chromatography on a Concanavalin A-Sepharose column (LKBInstruments) to remove a mannan containment that is contributed by theoriginal bacterial tryptic soy broth growth medium and that co-purifieswith the bacterial polysaccharide adhesin. The unbound fraction isrepeatedly dialyzed against water to remove salts and small molecules,then lyophilized. After reconstitution of the adhesin-containing powderin calcium-containing buffer at an acidic pH (preferably 5.0), thesolution is incubated sequentially with DNase (to remove contaminatingDNA), RNase (to remove contaminating RNA), and pronase (to removecontaminating protein). The purified adhesin solution is thenfractionated on a molecular sieve column in an ammonium carbonate bufferat neutral pH (preferably about 7.0). Elution is monitored by measuringA₂₀₆ nm; adhesin fractions eluting with a Kav of 0.0-0.2 are collectedand pooled. This fraction contains substantially pure capsularpolysaccharide adhesin.

Isolation of Teichoic Acid

Teichoic acid, another component of the slime of S. epidermidis, isrecovered from the DEAE Zeta Prep 250 ion-exchange column used infractionating adhesin, in the fraction eluting with a higherconcentration (0.6 M) of NaCl than eluted adhesin (0.2 M). This materialis then digested with nuclease enzymes as described above, protein isdenatured by heating at 100° C., at an acid pH (preferably about 4.0),then chromatographed on a molecular sieve column (Sepharose CL-4B) inammonium carbonate buffer at neutral pH. Serologically-active fractionsthat elute with a Kav of 0.33-0.57 are pooled, dialyzed, andlyophilized.

Adhesin Vaccine

Polyclonal Antibodies

Polyclonal antibodies to epitopic regions of the purified adhesin may beraised by a plurality of injections of said adhesin antigen into a hostanimal. In a preferred embodiment, antibodies are produced in rabbits bysubcutaneous administration of 0.5 mg of antigen in complete Freund'sadjuvant, followed 7 days later by intravenous injections three timesweekly with 0.5 mg of antigen in saline. The thrice weekly injectionsare performed for 3 consecutive weeks, and blood is then drawn 5 daysafter the last injection. Normal (pre-immune) serum is obtained in allcases.

Polyclonal antibodies to purified adhesin may also be raised in ratsgiven three 50 μg injections five days apart, with blood drawn 5 daysafter the final injection.

Polyclonal antibodies to whole cells of S. epidermidis strains areraised in rabbits as previously described (Pier, G. B., et al., J.Infect. Dis. 147:494-503 (1983)).

Monoclonal Antibodies

Monoclonal antibodies are immunoglobulins directed to specific epitopicregions on an antigen. Monoclonal antibodies against the substantiallypure polysaccharide adhesin of the invention can be produced by thehybridoma technology of Köhler and Milstein (Köhler, G., Science23:1281-6 (1986); Milstein, C., Science 21:1261-8 (1986)).

Briefly, the purified adhesin is used to once-prime or hyperimmunizeanimal donors of antibody-producing somatic B cells (e.g., lymphocytes).Lymph nodes and spleens of immunized animals are convenient sources.Although mouse and rat lymphocytes give a higher percentage of stablefusions with mouse myeloma lines, the use of rabbit, human and frogcells is also possible. In a preferred embodiment, hyperimmunized mousespleen cells are used to make the fused cell hybrids.

Specialized myeloma cell lines are available for use inhybridoma-producing fusion procedures (Kohler, G., et al., Eur. J.Immunol. 6:511-9 (1976); Schulman, M., et al., Nature 276:269-70(1978)). Methods for generating hybrids of anti-adhesin antibodyproducing spleen or lymph node cells and myeloma cells usually comprisemixing somatic cells with myeloma cells in a 10:1 proportion (though theproportion can vary from 20:1 to 1:1, respectively) in the presence ofan agent(s) that promotes fusion. It is preferred that the same speciesof animal is the source of both the somatic and myeloma cells. Fusionmethods have been described by Köhler and Milstein (Köhler, G., et al.,Nature 256:495-7 (1975); Eur. J. Immunol. 6:511-19 (1976)), in whichSendai virus is the fusion agent, and by Gefter (Gefter, S., et al.,Somatic Cell Genet. 3:231-6 (1977)), in which polyethylene glycol is thefusion agent. In a preferred embodiment, the method of Gefter et al. ismodified to include dimethylsulfoxide as an additional fusion agent.

Isolation of clones and antibody detection are carried out by standardtechniques. Fusion cell hybrids are selected by culturing the cells onmedia that support growth of hybridomas but prevent the growth ofunfused myeloma cells. (The unfused somatic cells do not maintainviability in in vitro cultures and hence do not pose a problem.) In apreferred embodiment, myeloma cells lacking hypoxanthinephosphoribosyltransferase (HPRT⁻) are used. These cells are selectedagainst in a hypoxanthine/aminopterin/thymidine (HAT) medium in whichhybridoma cells survive due to the HPRT⁺ genotype of the spleen cells,but unfused myeloma cells do not. Myeloma cells with different geneticdeficiencies that can be selected against in media supporting the growthof genotypically competent hybrids are also possible.

The detection of anti-adhesin antibody-producing hybrids can be achievedby any one of several standard assays, including ELISA and RIAtechniques that have been described in the literature (Kennet, R., etal., eds., Monoclonal Antibodies, Hybridomas: A New Dimension inBiological Analysis, Plenum, New York, 1980, pp. 376-84; Bryan, L. E.,et al., J. Clin. Microbiol. 18:276-82 (1983)).

Once the desired fused cell hybrids have been selected and cloned intoindividual anti-adhesin antibody-producing cell lines, each cell linemay be propagated in either of two standard ways: injection of thehybridoma into a histocompatible animal and recovery of the monoclonalantibodies in high concentration from the body fluids of the animal(e.g., serum or ascites fluid), or propagation in vitro in tissueculture, wherein the antibody in high concentration is recoverable fromthe culture medium.

Therapeutic Use of Anti-Adhesin Antibody

Monoclonal antibodies specific to epitopic regions on thecolonization-mediating adhesin, as well as the non-specific polyclonalantibodies described above, can be used clinically for the prevention ortreatment of diseases caused by pathogenic bacteria producing andbearing such adhesins. For example, polyclonal and monoclonal antibodiesspecific for the capsular polysaccharide adhesin of the presentinvention can be administered to any animal species for the preventionand/or treatment of infections due to pathogenic Staphylococcusepidermidis, e.g., those that colonize polymeric implanted medicaldevices and catheters. By the term “administer” is intended, for thepurpose of this invention, any method of treating an animal with asubstance, such as orally, intranasally, or parenterally (intravenously,intramuscularly, or subcutaneously). By the term “animal” is intendedany living creature that is subject to staphlococcal infection,including humans, farm animals, domestic animals, or zoological gardenanimals.’ The mode of administration of these antibodies is preferablyparenteral. The antibodies may be suspended or dissolved in any ofseveral suitable liquid vehicles and delivered to the patient by any oneof several parenteral means. In some instances, and particularly wherehuman treatment is involved, purification may be desired or requiredpursuant to government regulations. Provided the antibody is present ina pharmacologically effective dosage, other liquid compositions are alsopharmaceutically effective, including mixtures of antibodies and skimmilk and/or antibodies in aqueous salt solutions of serum albumin. Inhumans, the antibody may be preferably administered in parenteral form,though any compatible carrier may be used. Of course, the dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. Preferably, the dosage shouldresult in a concentration of at least about one Ag of specific antibodyper milliliter of blood.

Diagnostic Use of Anti-Adhesin Antibody

The adhesin-specific antibodies are also useful for medical and researchpurposes. For example, the antibodies can be used diagnostically todetect with great accuracy the presence of Staphylococcus epidermidisstrains among a general population of bacteria. Other applicationsinclude the use of adhesin-specific monoclonal antibodies in affinitychromatography systems for the purification of Staphylococcusepidermidis polysaccharide adhesin or in assay systems for thequantitative estimation of such adhesin.

Use of Purified Adhesin as a Probe

The purified capsular polysaccharide adhesin of the invention can beused in conjunction with the adherence assays described supra as a probein designing new polymeric materials to which coagulase-negativestaphlococci bacteria will not adhere. Such new polymers would beextremely beneficial to patients in whom catheters and other medicalprosthetic devices and shunts are employed and who now suffer from thenosocomial effects of such bacteria.

Having now described the invention in general term, the followingspecific examples will serve to illustrate more fully the nature of thepresent invention, without acting as a limitation upon its scope.

EXAMPLE I Isolation of Strain PR-62A Adhesin

Staphylococcus epidermidis RP-62A was grown in 15 l of tryptic soy brothin an LSL Biolaffite fermentor with aeration (0.5 l/min), stirring (200rpm), and maintenance of the pH at 7.2 by titration with 50% acetic acidand 5 NaOH. After 18 hr growth at 37° C., the pH was adjusted to 5.0with 50% acetic acid and the temperature of the culture raised to95-100° C. for 1 h. After cooling, the pH was adjusted to 6.8, theculture removed from the fermentor, and bacterial cells removed bycentrifugation. The supernatant was passed through a 0.5μ filter andthen concentrated to about 400 ml on a Pellicon ultrafiltration system(Millipore Corp., Bedford, Mass.) using membranes with a molecularweight cut-off of 10,000 dalton. The supernatant was then diluted with 2l deionized water and reconcentrated to 400 ml. This step was repeateduntil the pH of the solution was 6.8 and the conductivity was around 4.8millisiemens. A portion (¼) of the solution was then applied to a DEAEZeta-prep 250 cartridge (LKB Instruments, Rockville, Md.) previouslyequilibrated in 0.05 M Tris buffer, pH 6.8. After loading, the cartridgewas washed with 600 ml of 0.05 N Tris buffer and the eluate discarded.The adhesin was then recovered in the fraction eluting with 0.2 N NaClin 0.05 M Tris buffer, after preliminary assays determined that thismolarity of NaCl eluted material which inhibited the adherence of strainRP-62A to silastic catheter tubing (see infra). The 0.2 M NaCl eluatewas pooled, dialyzed against numerous changes of deionized water, andlyophilized. The material was then resuspended in 0.1 M sodium acetate,pH 6.0, at 25 mg/ml. and chromatographed on an affinity column ofConcanavalin A-Sepharose (LKB Instruments) to remove a mannan componentfrom the tryptic soy broth medium which co-purified with the adhesin.The unbound adhesin-containing fraction was recovered, dialyzed againstnumerous changes of deionized water, and lyophilized. The material wasthen dissolved (25 mg/ml) in 0.1 M NaOH, 1.0 mM MgCl₂, and 1.0 mMCaCl₂,pH 5.0, and digested with DNase (1 mg/ml) and RNase (3 mg/ml) for16 hr at 37° C., after which time pronase (1.0 mg/ml) was added and anadditional 4 hr digestion at 37° C. carried out. This solution was thenapplied to a 2.6×90 cm column of Sepharose CL-4B (Pharmacia FineChemicals, Piscataway, N.J.) equilibrated in 0.2 M ammonium carbonate,pH 6.8. Fractions (8 ml) were collected, and pools were made fromfractions absorbing UV light at 206 nm that eluted with a K_(av) of0.0-0.2 (peak=0.02).

EXAMPLE II Isolation of Strain PR-62A Teichoic Acid

Teichoic acid was recovered from the Zeta-prep 250 cartridge in thefraction eluting with 0.6 M NaCl. This material was digested withnuclease enzymes as described above, heated at 100° C., pH 4.0, for 1 h,then chromatographed on a 2.6×90 cm column of Sepharose CL-4B in 0.2 Mammonium carbonate. Serologically active fractions eluting with a Kav of0.33-0.57 (peak=0.48) were pooled, dialyzed against deionized water, andlyophilized.

EXAMPLE III Chemical Components of Crude Extract, Teichoic Acid Fractionof Slime, and Purified Adhesin

Utilizing the methodology described above, a fraction isolated from theculture supernatant of S. epidermidis strain RP-62A that appeared tohave the properties of an adhesin was analyzed. The chemical componentsof the crude extract, the isolated teichoic acid, and the purifiedadhesin are shown in Table 1.

TABLE 1 Chemical Components Identified in Crude Extract, Teichoic Acid,and Purified Adhesin of Staphylococcus epidermidis strain RP-62APreparation Component Crude Extract Teichoic Acid Purified AdhesinReducing sugar 12*   20    54    Amino sugars  5    25    20    Uronicacids  2    <1**  10    Phosphate 11    14    <0.02** Protein  3    2     1    Nucleic acids  7     1     1    Lipids <0.01** <0.01**<0.01**  Unidentified 60    38    14    Monosaccharides (percent oftotal sugars) Glycerol 20    <0.1**  Glucose 20    <0.1**  Galactose<0.1**  22    Glucosamine 10    15    Galactosamine <0.1**   5   *Percent of total weight. **Lower limit of detection.

Crude extract contained numerous components, of which carbohydrate andphosphate were predominant. The teichoic acid fraction of slime wascomposed principally of phosphate, glycerol, glucose, and glucosamine.The purified adhesin was principally composed of carbohydrate with onlylow to non-detectable levels of protein, nucleic acids, and phosphate.No lipids were detected in the purified adhesin. The principalmonosaccharides identified were galactose, glucosamine andgalactosamine, glucose was absent. In addition, a complex chromatogramof monosaccharides indicated the presence of galacturonic and glucuronicacids, as well as smaller amounts of mannosamine, fucosamine, andneuraminic acid. Trace amounts of ribose and muramic acid wereidentified, likely due to low level contamination with RNA andpeptidoglycan.

EXAMPLE IV Serological Properties of Crude Extract, Teichoic Acid, andPurified Adhesin

Serologically, crude extract gave three precipitin lines in doublediffusion when tested against a rabbit antisera raised against wholecells of strain RP-62A (FIG. 1A), while teichoic acid and the purifiedadhesin gave single precipitin lines. By immunoelectrophoresis (FIG.1B), the crude extract had multiple precipitin lines against antisera towhole cells. In contrast, purified adhesin gave a single precipitin linewhich did not move in the electric field. Purified teichoic acid gave astrong precipitin line migrating towards the anodal end of the gel, aswell as a weaker, more negatively charged line when high concentrationsof antigen were used. A mixture of teichoic acid and purified adhesinresulted in two precipitin lines corresponding to the individual,purified components.

EXAMPLE V Adherence of S. Epidermidis Strains to Polymeric Tubing

The adherence assay described supra was used to quantify the binding ofstrains of coagulase-negative staphylococci to silastic catheter tubing.When the inoculant size of strain RP-62A was varied from 10²-10⁹ cfu/ml,linear binding was obtained between input inocula of 10³-10⁶ cfu/ml.When 10⁶ cfu/ml of radiolabeled bacteria were used in this adherenceassay, and pieces of catheter tubing counted before and after beingrolled over the tryptic soy agar plate, 67-75% of the counts weredislodged in three separate experiments, indicating that a majority ofthe adherent bacterial population was being measured by this technique.

Strains of coagulase-negative staphylococci were screened in theadherence assay at inocula of 10⁶ cfu/ml. Three highly adherent strainsof coagulase-negative staphylococci in addition to strain RP-62A(strains RP-12, RP-14, F-3284), and poorly adherent strains (Table 2).

TABLE 2 Expression of Slime and Adhesin, and Adherence ofCoagulase-Negative Staphylococci to Silastic Catheter Tubing Productionof: Mean No. CFU Strain Species Slime¹ Adhesin Adhering (±SD) RP-62A S.epidermidis +++ Pos² 233 ± 20 RP-12 S. epidermidis +++ Neg 295 ± 40RP-14 S. hominis + Pos 167 ± 24 F-3284 S. epidermidis ++ Pos 144 ± 3 RP-62NA S. epidermidis − Pos³  68 ± 30 SP-2 S. haemolyticus − Neg  7 ± 7CL S. haemolyticus − Neg 19 ± 5 ¹Semi-quantitative measurement asdescribed by Christensen, G. D., et al. Infect. Immun. 37:318-26 (1982).²Presence (Pos) or absence (Neg) of adhesin determined by doubleimmunodiffusion. ³Strain RP-62NA is only weakly positive for adhesinproduction.

Of the three additional highly adherent strains, two expressed anantigen giving a precipitin line of identity in double diffusion withthe purified adhesin of strain RP-62A, while two of the poorly adherentstrains expressed no detectable antigen. The adherence properties ofstrain RP-62NA were also evaluated in the adherence assay (Table 2).Strain RP-62NA adhered only about ⅓ as well as its parent strain, and aweak precipitin line corresponding to purified adhesin could be detectedby ‘immunodiffusion only if culture super-natants of strain RP-62NA wereconcentrated 10-fold.

Restriction enzyme digestion of total cellular DNA of strains RP-62A andRP-62NA indicated that the parent strain and its variant were closelyrelated, as the digestion patterns using four different restrictionenzymes were identical (FIG. 2).

EXAMPLE VI Properties of Purified Adhesin

The adhesin purified from S. epidermidis strain RP-62A was tested forits ability to inhibit adherence of the homologous strain to silasticcatheter tubing. A dose-related inhibition of adherence was seen withboth crude extract and purified adhesin prepared from strain RP-62A(FIG. 3). Teichoic acid did not inhibit adherence of strain RP-62A, nordid the extract from the poorly adherent strain SP-2, prepared in amanner identical to that of crude extract from strain RP-62A. When thesesame materials (0.1 mg/ml) were tested for their ability to inhibitadherence of other strains of coagulase-negative staphylococci tosilastic catheter tubing, only the two strains expressing the adhesinantigen were significantly (P<0.05, t test) inhibited from adhering bypurified adhesin (FIG. 4). Some of the strains were inhibited fromadhering to the catheter material by crude extract from strain RP-62A,and adherence of strain SP-2 was inhibited by teichoic acid from strainRP-62A. In a similar fashion, rabbit antibodies raised to strainRP-62-A-purified adhesin from strain RP-62A inhibited the adherence ofthis strain in a dose-related fashion at serum concentrations of ≧0.25%.Using a serum concentration of 1%, there was significant (P<0.05, ttest) inhibition of adherence of strains of coagulase-negativestaphylococci expressing the adhesin antigen, while antigen-negativestrains were not inhibited from adhering to silastic catheter tubing atthis serum concentration (Table 3).

TABLE 3 Inhibition of Adherence of Coagulase-Negative Staphylococci toSilastic Catheter Tubing by Rabbit Antibody to Adhesin Purified fromStaphylococcus epidermidis Strain RP-62A Percent Inhibition of StrainAdherence (±1 SD)¹ Adhesin positive RP-62A 59 ± 17* RP-14 53 ± 1* F-3284 65 ± 14* RP-62NA²  1 ± 13  RP-12³ 62 ± 8  Adhesin negative SP-217 ± 9  CL 0 ¹Serum concentration 1%. ²p < 0.05, t test. *Producesgreatly reduced amount of adhesin. ³Initial studies with RP-12 werenegative. However, more recent studies have shown that RP-12 does infact produce this adhesin. Apparently, the sera used initially failed todetect the production of adhesin from strain RP-12.

Silastic catheter tubing coated with the purified adhesin readily boundrabbit antibodies raised to whole cells and purified adhesin, whileantibodies in pre-immunization sera had only a slight reaction withcoated catheter tubing (Table 4).

TABLE 4 Reaction of Rabbit Antibody to Purified Adhesin from StrainStaphylococcus epidermidis RP-62A with Silastic Catheter Tubing CatherCoated with Purified Adhesin Reaction with: Serum UncoatedAdhesin-Coated (5% Concentration) Catheter Catheter Pre-immune 0.150*0.202 Immune to Whole cell 0.191 1.212 Purified adhesin 0.076 1.443*Mean A₄₀₅ of triplicate wells containing indicated catheter.

EXAMPLE VII Transmission Electron Microscopy

Transmission electron microscopy was used to examine the appearance ofbacterial cells of RP-62A, RP-62NA, RP-14, RP-12 and CL after treatmentwith normal rabbit or rat serum, rabbit antiserum raised to whole RP-62Acells, and rat antiserum raised to purified adhesin. Both of theseantisera revealed an extracellular structure surrounding strains RP-62Aand RP-14 that appeared to be a capsule (FIGS. 5A-D) which was not seenwith normal serum (shown in the figure only for normal rabbit serum andstrain RP-62A; all other strains treated with any normal serum lookedidentical to FIG. 5A). Strain RP-62NA appeared to have only a slightamount of capsular material when reacted with antibodies to whole cells(FIG. 5F) and purified adhesin (not shown), consistent with theserologic findings mentioned supra. Both strains RP-12 and CL lacked anydetectable capsule using sera to RP-62A whole cells and purified adhesin(not shown).

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by LETTERS PATENT OF THE UNITED STATES:
 1. A method of producing monoclonal antibodies against a substantially pure coagulase-negative bacterial capsular polysaccharide adhesin of Staphylococcus epidermidis that binds to polymeric material comprising: propagating a hybridoma formed by fusing a cell capable of producing antibodies against said adhesin with a myeloma cell and harvesting the antibodies produced by said hybridoma, wherein said adhesin inhibits at a concentration of about 0.1 mg/ml, binding to said polymeric material of at least about 50% of cells of Staphylococcus epidermidis RP-62A, RP-62NA or F-3248.
 2. A composition comprising a monoclonal antibody that binds to a substantially-pure coagulase-negative bacterial capsular polysaccharide adhesin of Staphylococcus epidermidis that binds to polymeric material, wherein said adhesin inhibits at a concentration of about 0.1 mg/ml, binding to said polymeric material of at least about 50% of cells of Staphylococcus epidermidis RP-62A, RP-62NA or F-3284.
 3. A monoclonal antibody preparation comprising a monoclonal antibody that binds to an adhesin obtained from a Staphylococcus epidermidis RP-62A culture using the Isolation of Strain RP-62A Adhesin procedure comprising the following steps: (a) extracting adhesin from cultures of said bacterial strain RP-62A; (b) digesting the adhesin; (c) chromatographically separating said adhesin extract of (a) on an ion-exchange column; (d) eluting a capsular polysaccharide from the column of (c) which binds to silastic materials and inhibits binding of Staphylococcus epidermidis RP-62A, RP-62-NA, and F-3284 to said polymeric materials; (e) chromatographically separating the capsular polysaccharide adhesin-containing fractions from (d) on an affinity column; and (f) further purifying the capsular polysaccharide adhesin by chromatographing the adhesin-containing fractions from (e) on a molecular sieve column, and collecting material eluting between Kav of 0.0 to about 0.2.
 4. A pharmaceutical composition comprising the monoclonal antibody of claim 3 and a pharmaceutically compatible carrier.
 5. A hybridoma for producing the monoclonal antibody of claim 3 made by a process comprising the step of fusing: a) a cell capable of producing antibodies against the adhesin obtained from a Staphylococcus epidermidis RP-62A culture using the Isolation of Strain RP-62A Adhesin procedure comprising the following steps: (i) extracting adhesin from cultures of said bacterial strain RP-62A; (ii) digesting the adhesin; (iii) chromatographically separating said adhesin extract of (i) on an ion-exchange column; (iv) eluting a capsular polysaccharide from the column of (iii) which binds to silastic materials and inhibits binding of Staphylococcus epidermidis RP-62A, RP-62-NA, and F-3284 to said polymeric materials; (v) chromatographically separating the capsular polysaccharide adhesin-containing fractions from (iv) on an affinity column; and (vi) further purifying the capsular polysaccharide adhesin by chromatographing the adhesin-containing fractions from (v) on a molecular sieve column, and collecting material eluting between Kav of 0.0 to about 0.2, and b) a myeloma cell. 